Differentiated cell production method and culture bag used therefor

ABSTRACT

A method for producing differentiated cells from pluripotent stem cells by an embryoid body (EB) method, including the steps of: (1) culturing pluripotent stem cells by using a culture bag having a perfluoropolymer on its inner surface to thereby form an embryoid body; and (2) inducing differentiation of the pluripotent stem cells contained in the embryoid body obtained in step (1) to thereby obtain differentiated cells.

TECHNICAL FIELD

The present invention relates to a method for producing differentiatedcells and to a culture bag for use in the production method.

BACKGROUND ART

Pluripotent stem cells, which have the ability to differentiate intovarious cells, are expected to find clinical application or applicationto drug discovery research etc. Studies on using induced pluripotentstem cells (iPS cells) and embryonic stem cells (ES cells) aspluripotent stem cells are ongoing.

In use of pluripotent stem cells, a method for efficiently inducingdifferentiation into a desired differentiated cell is required. A methodfrequently used to induce differentiation of pluripotent stem cells isthe “embryoid body (EB) method,” which comprises culturing in alow-adherent culture dish (Petri dish) to allow for spontaneousaggregation of pluripotent stem cells, thereby forming an embryoid body(EB), which is a three-dimensional mass. The efficiency of inductioninto a desired cell species can be enhanced by adding cytokine, a growthfactor, or other compounds that have differentiation-inducing effects onculture of an embryoid body.

In the EB method, for example, a low-adherent Petri dish coated with ahydrogel or a hydrophilic polymer that has a phospholipid analogousstructure, such as 2-methacryloyloxyethyl phosphorylcholine, has beenused as a non-adherent culture dish (Petri dish) (PTL 1).

CITATION LIST Patent Literature

PTL 1: WO2005/001019

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a method that enablesan EB method to be carried out on a large scale in a closed system, ascompared with conventional methods using a culture dish.

Solution to Problem

To achieve the above object, the present inventors carried out extensiveresearch and arrived at the idea that bag culture, which enables alarger amount of pluripotent stem cells to be cultured in a suspension,would be advantageous over conventional culture using Petri dishes.However, the inventors found it difficult to produce a gas-permeableculture bag by using the same material as that of Petri dishesconventionally used for the EB method. The inventors further repeatedtrial and error and found that by using a culture bag having asufficiently gas-permeable and superhydrophobic perfluoropolymer on itsinner surface, the EB method can be carried out in a closed system on alarger scale than, and as efficiently as, conventional methods using aculture dish. The inventors thus achieved the present invention.

The present invention has been accomplished based on the above findings,and includes the following embodiments.

Item 1. A method for producing differentiated cells from pluripotentstem cells by an embryoid body (EB) method, comprising the steps of:(1) culturing pluripotent stem cells by using a culture bag having aperfluoropolymer on its inner surface to thereby form an embryoid body;and(2) inducing differentiation of the pluripotent stem cells contained inthe embryoid body obtained in step (1) to thereby obtain differentiatedcells.

Item 2.

A method for inducing differentiation of pluripotent stem cells by anembryoid body (EB) method, comprising the steps of:

(1) culturing pluripotent stem cells by using a culture bag having aperfluoropolymer on its inner surface to thereby form an embryoid body;and(2) inducing differentiation of the pluripotent stem cells contained inthe embryoid body obtained in step (1) to thereby obtain differentiatedcells.

Item 3.

The method according to Item 1 or 2, wherein the perfluoropolymer is atleast one perfluoropolymer selected from the group consisting oftetrafluoroethylene-hexafluoropropylene copolymers,tetrafluoroethylene-perfluoroalkyl vinyl ether copolymers, andtetrafluoroethylene-hexafluoropropylene-perfluoroalkyl vinyl ethercopolymers.

Item 4.

The method according to any one of Items 1 to 3, wherein the pluripotentstem cells are induced pluripotent stem cells (iPS cells) or embryonicstem cells (ES cells).

Item 5.

The method according to any one of Items 1 to 4, wherein in step (2),the pluripotent stem cells are differentiated into progenitor cells.

Item 6.

A culture bag having a perfluoropolymer on its inner surface, the bagbeing for use in inducing differentiation of pluripotent stem cells byan embryoid body (EB) method.

Item 7.

The culture bag according to Item 6, wherein the perfluoropolymer is atleast one perfluoropolymer selected from the group consisting oftetrafluoroethylene-hexafluoropropylene copolymers,tetrafluoroethylene-perfluoroalkyl vinyl ether copolymers, andtetrafluoroethylene-hexafluoropropylene-perfluoroalkyl vinyl ethercopolymers.

Item 8.

The culture bag according to Item 6 or 7, wherein the pluripotent stemcells are induced pluripotent stem cells (IPS cells) or embryonic stemcells (ES cells).

Item 9.

An embryoid body that is obtainable by step (1) according to any one ofItems 1 to 4.

Item 10.

A differentiated cell population that is obtainable by the productionmethod according to any one of Items 1 and 3 to 5.

Advantageous Effects of Invention

According to the present invention, an EB method can be carried out on alarger scale than conventional methods using a culture dish.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates an example of the culture bag of the presentinvention.

FIG. 2 shows results of the Example.

FIG. 3 shows results of the Example.

FIG. 4 shows results of the Example.

FIG. 5 shows results of the Example.

FIG. 6 shows results of the Example.

FIG. 7 shows results of the Example.

FIG. 8 shows results of the Example.

FIG. 9 shows results of the Example.

FIG. 10 shows results of the Example.

FIG. 11 shows results of the Example.

FIG. 12 shows results of the Example.

FIG. 13 shows results of the Example.

FIG. 14 snows results of the Example.

FIG. 15 shows results of the Example.

FIG. 16 shows results of the Example.

FIG. 17 shows results of the Example.

FIG. 18 shows results of the Example.

FIG. 19 shows results of the Example.

FIG. 20 shows results of the Example.

FIG. 21 snows results of the Example.

FIG. 22 shows results of the Example.

FIG. 22 shows results of the Example.

FIG. 23 shows results of the Example.

FIG. 24 shows results of the Example.

FIG. 25 shows results of the Example.

FIG. 26 shows results of the Example.

FIG. 27 shows results of the Example.

FIG. 28 shows results of the Example.

FIG. 29 shows results of the Example.

FIG. 30 shows results of the Example.

DESCRIPTION OF EMBODIMENTS 1. Method for Producing Differentiated Cellsand Culture Bag for Use in the Method

The method for producing differentiated cells of the present inventionis a method for producing differentiated cells from pluripotent stemcells by an embryoid body (EB) method, the method comprising the stepsof:

(1) culturing pluripotent stem cells by using a culture bag having aperfluoropolymer on its inner surface to thereby form an embryoid body;and(2) inducing differentiation of the pluripotent stem cells contained inthe embryoid body obtained in step (1).

1.1 Step (1) 1.1.1 Culture Bag

The culture bag comprises a perfluoropolymer on its inner surface. Thesurface with which the culture cells may come into contact during theculture is covered with at least a perfluoropolymer. This preventspluripotent stem cells from adhering to the surface of the culture bag,whereby an embryoid body can be formed normally.

The culture bag may be formed of a sufficiently gas-permeableperfluoropolymer itself. In that case, the film that forms the culturebag is composed of a perfluoropolymer itself and may optionally furtherhave one or more coatings, if necessary.

To obtain the effect of the present invention, the perfluoropolymer mustbe present in the portion with which cells come into contact. As long asthis condition is met, such coatings can be optionally applied.Accordingly, except for the case in which there are specialcircumstances, such coatings, if used, are usually applied to theoutside of the culture bag or portions with which cells do not come intocontact.

The size of the culture bag is not limited and can be suitably set. Thelower limit of the capacity of the culture bag may be, for example, butis not limited to, 10 ml, 20 ml, 50 ml, or 100 ml. The upper limit ofthe capacity may be, for example, but is not limited to, 500 ml, 400 ml,300 ml, or 200 ml. The capacity range of the culture bag may be, forexample, 10 ml to 500 ml.

In this specification, “perfluoropolymer” refers to a polymer thatbasically does not contain hydrogen in the molecule and that is composedof carbon and fluorine, or a polymer that is basically composed ofcarbon and fluorine and that partially contains oxygen etc.

The perfluoropolymer optionally contains a non-fluorinated groupterminal. In the present invention, “non-fluorinated group terminal”refers to an end that is reactive and is generally called an “unstableend.” Specific examples include functional groups, such as —COF, —COOH,—COOH associated with water, —CH₂OH, —CONH₂ and —COOCH₃. Suchnon-fluorinated group terminals are formed by being derived from, forexample, a reaction initiator in a polymerization reaction.

The perfluoropolymer is preferably a perfluoropolymer in which the totalnumber of non-fluorinated group terminals is 70 or less per 1×10⁶ carbonatoms. In this case, high non-adherence to cells can be exhibited. In amore specific embodiment of the present invention, the perfluoropolymeris preferably a perfluoropolymer in which the total number of —COF,—COOH, —COOH associated with water, —CH₂OH, —CONH₂ and —COOCH₃ ispreferably 70 or less per 1×10⁶ carbon atoms.

The total number of the non-fluorinated group terminals per 1×10⁶ carbonatoms is preferably, with increasing preference in the order given, 50or less, 35 or less, 15 or less, 10 or less, 5 or less, or 2 or less.The same applies to the total number of —COF, —COOH, —COOH associatedwith water, —CH₂OH, —CONH₂ and —COOCH₃.

The perfluoropolymer is preferably a perfluoropolymer in which the totalnumber of non-fluorinated group terminals and —CF₂H group terminals is70 or less per 1×10⁶ carbon atoms. The total number of thenon-fluorinated group terminals and —CF₂H group terminals per 1×10⁶carbon atoms is preferably, with increasing preference in the ordergiven, 35 or less, 20 or less, or 10 or less.

Preferably, the perfluoropolymer does not contain —CF₂H group terminals.

The number of the non-fluorinated group terminals and —CF₂H groupterminals can be calculated by FT-IR.

The method for adjusting the amount of non-fluorinated groups in theperfluoropolymer to the range mentioned above may be, for example, butis not limited to, the following methods: a method of controllingterminal groups by using a chain transfer agent or a polymerizationcatalyst in a polymerization reaction to thereby inhibit creation ofnon-fluorinated group terminals; a method of fluorinatingnon-fluorinated group terminals by bringing a polymer obtained by apolymerization reaction into contact with a fluorine-containingcompound, which functions as a fluorine radical source; and the like.

In the above, the operation of bringing the perfluoropolymer obtained bythe polymerization reaction into contact with a fluorine-containingcompound, which functions as a fluorine radical source, can also beperformed at any stage before or after melt extrusion after aperfluoropolymer is obtained by a method such as suspensionpolymerization or emulsion polymerization. This operation can also beperformed at a stage after shaping a perfluoropolymer. This operationcan be effectively performed by repeating the operation at two or moreof the following three stages: before and after melt extrusion, andafter shaping.

The fluorine-containing compound that functions as a fluorine radicalsource and that is used in the method of fluorinating non-fluorinatedgroup terminals is not particularly limited and can be selected from awide variety of fluorine-containing compounds. Examples includefluorinated halogens, such as IF₅ and ClF₃, fluorine gas (F₂), COF₃,AgF₂, UF₆, OF₂, N₂F₂, CF₃OF, and the like.

When fluorine gas is used, 100% fluorine gas may be used. However,mixing fluorine gas with an inert gas and then using the mixture isrecommendable in terms of safety. In that case, the mixing ratio may be,for example, but is not limited to, a fluorine gas concentration of 5 to50 mass %. A fluorine gas concentration of 15 to 30 mass % isparticularly preferable. The inert gas to be used is not limited, andmay be selected from a wide variety of gases, such as nitrogen gas,helium gas, and argon gas. Nitrogen gas is preferable in terms of costperformance.

In the method of fluorinating non-fluorinated group terminals comprisingbringing the polymer obtained by a polymerization reaction into contactwith a fluorine-containing compound, which functions as a fluorineradical source, the processing temperature is preferably 20 to 220° C.,and more preferably 100 to 200° C. The processing time is preferably 5to 30 hours, and more preferably 10 to 20 hours.

The perfluoropolymer is preferably a homopolymer or a copolymercomprising repeating units derived from at least one perfluoroethylenicmonomer.

The perfluoropolymer preferably comprises repeating units derived fromat least one fluoroethylenic monomer selected from the group consistingof tetrafluoroethylene (TFE), hexafluoropropylene (HFP), andperfluoro(alkyl vinyl ether) (PAVE) represented by CF₂═CF—ORf (whereinRf is a perfluoroalkyl group having 1 to 8 carbon atoms).

Examples of PAW, include, but are not limited to, perfluoro(methyl vinylether) (FMVE), perfluoro(ethyl vinyl ether) (PEVE), perfluoro(propylvinyl ether) (PPVE), perfluoro(butyl vinyl ether), perfluoro(pentylvinyl ether), perfluoro(hexyl vinyl ether), perfluoro(heptyl vinylether), and the like.

Examples of perfluoropolymers include polytetrafluoroethylenes (PTFE),TFE-HFP copolymers (FEP), TFE-PAVE copolymers (PFA), TFE-HFP-PAVEcopolymers, and the like. Among these, FEP and PFA are preferable, andFEP is more preferable.

The mass ratio of TFE:HFP in FEP is preferably in the range of 80:20 to97:3, and more preferably 84:16 to 92:8.

The mass ratio of TFE:PAVE in PFA is preferably in the range of 90:10 to98:2, and more preferably 92:8 to 97:3.

The mass ratio of TFE:HFP:PAVE in the TFE-HFP-PAVE copolymer ispreferably 70 to 97:2.5 to 20:0.1 to 10.

The culture bag can be produced, for example, by superposing two sheetsof film comprising a perfluoropolymer as described above and thenheat-sealing the edge portions by laser joining or using an impulsesealer, or the like.

1.1.2 Pluripotent Stem Cells

The pluripotent stem cells to be used are not limited and may be, forexample, undifferentiated cells that have both self-replication abilityand differentiation pluripotency. Self-replication ability means thatcells can proliferate while maintaining an undifferentiated state.Differentiation pluripotency means that cells can differentiate into allthree germ layer lineages.

Specific examples of pluripotent stem cells include ES cells, iPS cells,embryonal carcinoma (EC) cells, which are derived from teratocarcinomacells, embryonic germ (EG) cells, which are derived from primordial germcells, multipotent germline stem (mGS) cells, which can be isolatedduring the process of establishing and culturing GS cells fromtesticular tissue, and multipotent adult progenitor cells (MAPC), whichcan be isolated from bone marrow.

Examples of usable ES cells include ES cells produced by nuclearreprogramming of somatic cells.

Examples of pluripotent stem cells that can be preferably used includeES cells and iPS cells.

The origin of pluripotent stem cells is not limited, and can be selectedfrom mammals according to the purpose of use of the desireddifferentiated cells. As long as pluripotent stem cells can beestablished, the origin of the cells can be selected, for example, fromhumans, monkeys, pigs, rabbits, dogs, rats, mice, etc.

The origin of iPS cells is not particularly limited, and can beappropriately selected from various somatic cells. Examples includefibroblasts, synoviocytes, T lymphocytes, dental pulp stem cells,umbilical cord blood cells, peripheral mononuclear blood cells, and thelike.

The concentration of pluripotent stem cells in the culture is notlimited, and can be appropriately set according to, for example, thekind of cells used. The concentration may be, for example, 3.3×10³ to3.3×10⁵ cells/ml, preferably 3.3×10⁴ to 3.3×10⁵ cells/ml, and morepreferably about 3.3×10⁴ cells/ml.

1.1.3 Culture Conditions

The culture conditions are not particularly limited, and can follow theconditions used in a usual EB method. The culture conditions may beoptionally modified appropriately.

The process up to the formation of an embryoid body can be summarized asfollows. Pluripotent stem cells are dispersed in a culture liquid, whichmay contain various additives, such as serum and a growth factor, ifnecessary, to thereby obtain a cell suspension. The cell suspension isplaced in the culture container of the present invention and cultured ina predetermined environment, such as in a CO₂ incubator. An embryoidbody is usually formed in about 2 to 7 days after initiating theculture.

The culture is preferably performed while shaking the culture bag. Theshaking conditions are not limited, and can be appropriately set.

1.2 Step (2)

Step (2) is a step of obtaining differentiated cells by inducingdifferentiation of pluripotent stem cells contained in the embryoid bodyobtained in step (1) described above.

The conditions for inducing the differentiation are not limited, and canbe appropriately set according to, for example, the kind of cell usedand the kind of desired differentiated cell. The differentiation can beusually induced by adding a specific cytokine, growth factor, or othercompounds to a culture liquid in predetermined concentrations, andculturing.

Steps (1) and (2) may be performed simultaneously. In this case, step(1) can be performed in the presence of the compound used in step (2).

The differentiated cells are not limited, and can be any of variousprogenitor cells. For example, hematopoietic progenitor cells can beobtained by using T lymphocyte-derived T-iPS cells as pluripotent stemcells and performing steps (1) and (2) under predetermined conditions.

2. Embryoid Body and Differentiated Cell Population

The embryoid body of the present invention can be obtained by step (1)in the production method of the present invention.

The embryoid body of the present invention is considered to have atleast properties similar to those of an embryoid body obtained by theconventional EB method.

The differentiated cell population is a population of differentiatedcells obtained by the production method of the present invention.

EXAMPLES

Experimental Examples (including Examples and Comparative Examples) aregiven below to further clarify the structural features and effects ofthe present invention. The Experimental Examples are merely examples tofacilitate understanding of the present invention. The scope of theinvention is not limited by the Experimental Examples.

Examples 1 to 4: Production of Culture Bags

Pellets of FEP and PFA, individually used as materials, were melted andshaped to thereby form different types of film.

Two types of film were prepared in the same manner as above except thatthe pellets were fluorinated when melt-extruded.

Each type of film with a size of 106 mm×66 mm and a thickness of 100 μmwas heat-sealed using an impulse sealer under the following conditions:with a seal width of 3 mm, at a sealing pressure of 0.2 MPa, for asealing time of 50 seconds. One port made of the same material as eachfilm was attached to an upper portion of the heat-sealed film to therebyform four kinds of bags (Examples 1 to 4) (FIG. 1).

A sample of each material with a thickness of about 250 to 300 μm wasformed by superposing the same type of film on one another. The samplesthus obtained were analyzed with an FT-IR Spectrometer 1760× (producedby Perkin-Elmer Inc.).

The samples were prepared by superposing the same type of film.

A differential spectrum between a reference (a sample fluorinated enoughuntil substantially no difference in spectra was able to be seen) andeach sample was obtained. Absorbance of each peak was read and thenumber of non-fluorinated group terminals and the number of —CF₂H groupterminals per 1*10⁶ carbon atoms were calculated according to thefollowing equation. Table 2 shows the number of non-fluorinated groupterminals and the number of —CF₂H group terminals in each culture bag.

The total number of non-fluorinated group terminals and —CF₂H groupterminals (per 1×10⁶ carbon atoms)=I·k/t

I: Absorbance

K: Correction coefficient (Table 1)t: Thickness of film (mm)

TABLE 1 Absorption wavenumber Correction Terminal group (cm⁻¹)coefficient —COF 1884 405 —COOH (not associated with water) 1813 455═COOH (associated with water) 1775 455 1790 —COOCH₃ 1795 355 —CONH₂ 3438480 —CH₂OH 3648 2325 —CH₂H 3006 26485

TABLE 2 Fluorine Number of non-fluorinated Number of —CF₂H resin groupterminals group terminals Example 1 FEP 21 424 Example 2 FEP 13 0Example 3 PFA 201 159 Example 4 PFA 25 0

Example 5: Production of Differentiation-induced Cell Population 1)Preparation of EB

T-iPS cells were cultured on a plastic plate (BD Culturedundifferentiated T-iPS cells were treated with TrypLE (GIBCO), and thecells were gently dissociated. The T-iPS cells were obtained by themethod disclosed in a known publication (Nishimura et al., “Generationof Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotencyand Redifferentiation,” Cell Stem Cell, 2013, 12, pp. 114-126).

A cell aggregate was resuspended in StemPro-34 (Invitrogen) containingpenicillin-streptomycin (10 ng/mL), L-glutamine (2 mM), ascorbic acid (1mM), monothioglycerol (MTG, 4×10-4 M; Sigma), transferrin (150 η/mL),and BMP-4 (osteogenic protein-4) (10 ng/mL).

15 ml of the cell suspension was placed in each of the bags obtained inExamples 1 to 4 and cultured. The number of cells on initiation of theculture was set to 5×10⁵⁻/bag and 5×10⁶⁻/bag.

After 24 hours, bPGF (a basic fibroblast growth (proliferation) factor)was added to a final concentration of 5 ng/ml, and the culture wascontinued. As a result, formation of EB was observed in all of the bags(FIG. 2).

On day 14 after initiation of the culture, the cells were collected, anddifferentiation tendency was analyzed using FACS. FACSAria™II, producedby BD Biosciences, was used as a flow cytometer.

For this analysis, fluorescently labeled antibodies against CD34(fluorescently labeled with APC), CD34 (fluorescently labeled withPacific Blue), CD43 (fluorescently labeled with PE), CD-14(fluorescently labeled with PE-Cy7), and CD235a (fluorescently labeledwith APC) were used.

The analysis results are in FIGS. 3 and 4 (Example 1; cell count: 5×10⁵cells), FIGS. 5 and 6 (Example 1; number of cells: 5×10⁶ cells), FIG. 7and 8 (Example 2; number of cells: 5×10⁵ cells), FIGS. 9 and 10 (Example2; number of cells: 5×10⁶ cells), FIG. 11 and 12 (Example 3; number ofcells: 5×10⁵ cells), FIG. 13 and 14 (Example 3; number of cells: 5×10⁶cells), FIG. 15 and 16 (Example 4; number of cells: 5×10⁵ cells), andFIG. 17 and 18 (Example 4; number of cells: 5×10⁶ cells).

Monocyte lineage cells (CD14⁺), erythroid cells (CD235a⁺), etc. wereobserved.

After differentiation was induced using the culture bag obtained inExample 1, the number of CD34⁺/CD43⁺ hematopoietic progenitor cells,which are fractionated using CD34 antibody and CD43 antibody, wasdetermined. When the number of cells on initiation of the culture was5×10⁵⁻/bag, 10,401 hematopoietic progenitor cells were obtained. Whenthe number of cells on initiation of the culture was 5×10⁶⁻/bag, 3,836hematopoietic progenitor cells were obtained.

Further, after differentiation was induced using the culture bagsobtained in Examples 3 and 4, the number of CD34⁺/CD43⁺ hematopoieticprogenitor cells, which were fractionated in the same manner as above,was determined. The results are in Table 3.

TABLE 3 5 × 10⁵ bag 5 × 10⁶ bag Culture bag of Example 3 9,409 cells6,479 cells Culture bag of Example 4 9,202 cells 6,425 cells

These results confirmed that differentiation of T-iPS cells intohematopoietic progenitor cells can be efficiently induced on a largescale regardless of the kind of culture bag used.

When the number of cells on initiation of the culture was 5×10⁵⁻/bag, ahigher yield, of hematopoietic progenitor cells was obtained as comparedwith the results obtained with the number of cells on initiation of theculture being 5×10⁶⁻/bag.

(2) Induction of Differentiation to T cells

The differentiation induction ability of CD34⁺ cells, which wereobtained by sorting in the above experiment, into T cells was confirmedby the method disclosed in the above-mentioned known document, Nishimuraet al.

The cells obtained from the four bags were collected and mixed. Theresulting mixture was transferred to gamma-irradiated OP9-DL1 cells(provided, by Riken BioResource Research Center; Watarai H. et al.,“Generation of functional MKT cells in vitro from embryonic stem cellsbearing rearranged invariant Val4-Ja18 TCRa gene,” 2010, Blood, 115, pp.230-237), and OP9medium (αMEM medium containing 15% fetal bovine serum,2 mM L-glutamine, 100 U/ml penicillin, and 100 ng/ml streptomycin) wasadded. These cells were co-cultured in the presence of FLT-3L(EMS-related tyrosine kinase 3 ligand) and IL-7 (interleukin 7) toinduce differentiation of T cell lines. Differentiation tendency wasanalyzed in the same manner as above using FACS. The culture wasperformed in an atmosphere containing 5% CO₂.

For this analysis, fluorescently labeled antibodies against CD45(fluorescently labeled with brilliant violet 510), TCRab (fluorescentlylabeled with FITC), CD3 (fluorescently labeled with APC-Cy7), CD7(fluorescently labeled with APC), CD5 (fluorescently labeled withPE-Cy7), CD4 (fluorescently labeled with brilliant violet 421), CD8α(fluorescently labeled with PerCP-Cy5.5), and CD8β (fluorescentlylabeled with PE) were used.

FIGS. 19 to 30 show the analysis results.

FIGS. 19 to 24 also show the results obtained by analyzing peripheralblood as a control in the same manner. A population of cells that wereintermediate in differentiation (CD4⁺/CD8⁺) was also confirmed in thepopulation of cells obtained by inducing differentiation of cellsobtained from the bag (on day 30) (FIGS. 25 to 30). The CD4 expressionlevel and CDS expression level were each equivalent to the expressionlevel of peripheral blood T cells. This result confirmed thathematopoietic progenitor cells obtained by induction in the bag also hadability to differentiate into T cells with very high efficiency.

DESCRIPTION OF THE REFERENCE NUMERALS 1: Culture bag 11: Port

12: Sealing part

1. A method for producing differentiated cells from pluripotent stemcells by an embryoid body (EB) method, comprising the steps of: (1)culturing pluripotent stem cells by using a culture bag having aperfluoropolymer on its inner surface to thereby form an embryoid body;and (2) inducing differentiation of the pluripotent stem cells containedin the embryoid body obtained in step (1) to thereby obtaindifferentiated cells.
 2. A method for inducing differentiation ofpluripotent stem cells by an embryoid body (EB) method, comprising thesteps of: (1) culturing pluripotent stem cells by using a culture baghaving a perfluoropolymer on its inner surface to thereby form anembryoid body; and (2) inducing differentiation of the pluripotent stemcells contained in the embryoid body obtained in step (1).
 3. The methodaccording to claim 1, wherein the perfluoropolymer is at least oneperfluoropolymer selected from the group consisting oftetrafluoroethylene-hexafluoropropylene copolymers,tetrafluoroethylene-perfluoroalkyl vinyl ether copolymers, andtetrafluoroethylene-hexafluoropropylene-perfluoroalkyl vinyl ethercopolymers.
 4. The method according to claim 1, wherein the pluripotentstem cells are induced pluripotent stem cells (iPS cells) or embryonicstem cells (ES cells).
 5. The method according to claim 1, wherein instep (2), the pluripotent stem cells are differentiated into progenitorcells. cm
 6. A culture bag having a perfluoropolymer on its innersurface, the bag being for use in inducing differentiation ofpluripotent stem cells by an embryoid body (EB) method.
 7. The culturebag according to claim 6, wherein the perfluoropolymer is at least oneperfluoropolymer selected from the group consisting oftetrafluoroethylene-hexafluoropropylene copolymers,tetrafluoroethylene-perfluoroalkyl vinyl ether copolymers, andtetrafluoroethylene-hexafluoropropylene-perfluoroalkyl vinyl ethercopolymers.
 8. The culture bag according to claim 6, wherein thepluripotent stem cells are induced pluripotent stem cells (iPS cells) orembryonic stem cells (ES cells).